This invention disclosure provides novel methods for measuring the activity, and modulation of the activity, of DNA topoisomerases, essential enzymes that control the topological state of DNA in cells. The methods of this invention are also useful for measuring the activity of other enzymes, which affect the topological state of DNA, which for example includes, but is not limited to, restriction enzymes.
In prokaryotes topoisomerases are targets of antibacterial agents. In eukaryotes they are anti-tumour drug targets and potential herbicide targets. All topoisomerases can relax supercoiled DNA, and DNA gyrase, present in bacteria, can also introduce supercoils into DNA. Despite being the target of some of the key anti-microbials and anti-cancer drugs in use today (e.g. ciprofloxacin, camptothecins), the basic reaction catalyzed by these enzymes, the inter-conversion of relaxed and supercoiled DNA, is not readily monitored.
The standard assay for monitoring the superhelical state of nucleic acids is an electrophoresis gel-based assay, which suffers from the drawback of being slow and, due to the electrophoresis step, requires a lot of sample handling.
In response to this limitation, efforts have been made in recent years to develop high-throughput assays for topoisomerases. Reference is made here, for example, to U.S. Pat. No. 5,998,152, issued on Dec. 7, 1999, and U.S. Pat. No. 6,197,527, issued on Mar. 6, 2001, both issued to Tularik, and both of which are hereby incorporated by reference for the purpose of defining the background and state of the art defined therein. In the methodology according to those patents, a topoisomerase-nucleic acid complex is formed, denatured and identified, either in a solid-phase or liquid-phase format. Stabilization of a covalent complex between the protein and the DNA, and, in the solid-phase mode, immobilization of the enzyme, is required. In the liquid-phase assay, the signal is FRET between two labels on the DNA. That form of the Tularik assay nonetheless still relies on formation of a covalent cleavage complex, and, in this case, separation of the labels following cleavage. Thus, a limiting feature of the Tularik methodology is the requirement that a cleavable-complex, stabilized by a potential drug, must be formed. As such, these known assays are limited in their ability to identify only one mode of enzyme inhibitors. For example, such an assay would potentially identify a quinolone, but would not identify an aminocoumarin, such as novobiocin. Although the quinolone-type drugs are the most successful anti-topoisomerase agents currently available, it is not at all clear that appropriate non-quinolone inhibitors may not be just as effective, if not more so, were it possible to readily identify them. Accordingly, this is a drawback for the known Tularik high-throughput screening assays.
Accordingly, those skilled in the art will appreciate that there remains an interest in developing novel assays (such as high-throughput assays) to measure enzyme activities. Preferred assays would be generally applicable to identification of compounds with relevant topoisomerase or gyrase modulating efficacy. Such methodology would greatly facilitate work on topoisomerases (and other enzymes), and would specifically potentiate the use of combinatorial chemical libraries to screen for novel lead compounds (antibiotics, anti-tumour drugs, herbicides).